Weijia Zhu

Bcl-2 mutants with restricted subcellular location reveal spatially distinct pathways for apoptosis in different cell types.

Human Bcl‐2 is located in multiple intracellular membranes when expressed in MDCK and Rat‐1/myc cells. We restricted expression to the endoplasmic reticulum or mitochondria by exchanging the Bcl‐2 carboxy‐terminal insertion sequence for an equivalent sequence from cytochrome b5 or ActA, respectively. MDCK cells are protected from serum deprivation‐induced apoptosis by both wild‐type Bcl‐2 and the mutant targeted to mitochondria but not by the mutant targeted to endoplasmic reticulum. In contrast, when expressed in Rat‐1/myc cells, the Bcl‐2 mutant located at the endoplasmic reticulum is more effective than that targeted to mitochondria. In MDCK cells both mutants bind Bax as effectively as wild‐type, demonstrating that Bax binding is not sufficient to prevent apoptosis.

Bcl‐2 changes conformation to inhibit Bax oligomerization

Bcl‐2 inhibits apoptosis by regulating the release of cytochrome c and other proteins from mitochondria. Oligomerization of Bax promotes cell death by permeabilizing the outer mitochondrial membrane. In transfected cells and isolated mitochondria, Bcl‐2, but not the inactive point mutants Bcl‐2‐G145A and Bcl‐2‐V159D, undergoes a conformation change in the mitochondrial membrane in response to apoptotic agonists such as tBid and Bax. A mutant Bcl‐2 with two cysteines introduced at positions predicted to result in a disulfide bond that would inhibit the mobility of α5–α6 helices (Bcl‐2‐S105C/E152C) was only active in a reducing environment. Thus, Bcl‐2 must change the conformation to inhibit tBid‐induced oligomerization of integral membrane Bax monomers and small oligomers. The conformationally changed Bcl‐2 sequesters the integral membrane form of Bax. If Bax is in excess, apoptosis resumes as Bcl‐2 is consumed by the conformational change and in complexes with Bax. Thus, Bcl‐2 functions as an inhibitor of mitochondrial permeabilization by changing conformation in the mitochondrial membrane to bind membrane‐inserted Bax monomers and prevent productive oligomerization of Bax.

Endoplasmic reticulum localized Bcl-2 prevents apoptosis when redistribution of cytochrome c is a late event

The disruption of mitochondrial function is a key component of apoptosis in most cell types. Localization of Bcl-2 to the outer mitochondrial and endoplasmic reticulum membranes is consistent with a role in the inhibition of many forms of apoptosis. In Rat-1 cells, a Bcl-2 mutant targeted exclusively to the endoplasmic reticulum (Bcl-cb5) was effective at inhibiting apoptosis induced by serum starvation/myc, or ceramide but not apoptosis induced by etoposide. The former conditions cause a decrease in mitochondrial transmembrane potential (Δψm) as an early event that precedes the release of cytochrome c from mitochondria. By contrast, when cells are exposed to etoposide, a situation in which cytochrome c release and membrane localization of the pro-apoptotic protein Bax precede loss of Δψm, wild type Bcl-2 but not Bcl-cb5 prevents apoptosis. Therefore, Bcl-2 functions in spatially distinct pathways of apoptosis distinguished by the order of cytochrome c release and loss of Δψm.

Cytoplasmic O-glycosylation prevents cell surface transport of E-cadherin during apoptosis

Cellular adhesion is regulated by members of the cadherin family of adhesion receptors and their cytoplasmic adaptor proteins, the catenins. Adhesion complexes are regulated by recycling from the plasma membrane and proteolysis during apoptosis. We report that in MCF‐7, MDA‐MB‐468 and MDCK cells, induction of apoptosis by agents that cause endoplasmic reticulum (ER) stress results in O‐glycosylation of both β‐catenin and the E‐cadherin cytoplasmic domain. O‐glycosylation of newly synthesized E‐cadherin blocks cell surface transport, resulting in reduced intercellular adhesion. O‐glycosylated E‐cadherin still binds to β‐ and γ‐catenin, but not to p120‐catenin. Although O‐glycosylation can be inhibited with caspase inhibitors, cleavage of caspases associated with the ER or Golgi complex does not correlate with E‐cadherin O‐glycosylation. However, agents that induce apoptosis via mitochondria do not lead to E‐cadherin O‐glycosylation, and decrease adhesion more slowly. In MCF‐7 cells, this is due to degradation of E‐cadherin concomitant with cleavage of caspase‐7 and its substrate poly(ADP‐ribose) polymerase. We conclude that cytoplasmic O‐glycosylation is a novel, rapid mechanism for regulating cell surface transport exploited to down‐regulate adhesion in some but not all apoptosis pathways.

Bcl-XL is qualitatively different from and ten times more effective than Bcl-2 when expressed in a breast cancer cell line

BackgroundBcl-2 and Bcl-XL are anti-apoptotic paralogues that inhibit apoptosis elicited by a wide variety of stimuli, and play critical roles in cancer development and resistance to treatment. Many clinical studies have indicated that expression of these anti-apoptotic proteins in tumours is associated with poor prognosis. It has therefore been assumed that in cells the essential difference between Bcl-2 and Bcl-XL involves regulation of expression and that they are otherwise functionally similar. To examine this issue, we have compared the function of the proteins and of mutants of Bcl-2 and Bcl-XL specifically targeted to different subcellular sites.MethodsWe generated clones of the human breast cancer line MCF-7 stably expressing known amounts of Bcl-2, or Bcl-XL as determined by quantitative immunoblotting. Clones expressing equivalent amounts of wild-type and mutants of Bcl-2 and Bcl-XL with subcellular localization restricted to the cytoplasm, endoplasmic reticulum or outer mitochondrial membrane were studied in both MCF-7 and Rat-1 fibroblasts. In MCF-7 cells we measured the functional activities of these proteins in preventing apoptosis induced by four different agents (doxorubicin, ceramide, thapsigargin, TNF-α). Etoposide and low serum were used to compare the effect of Bcl-2, Bcl-XL and mutants located at the endoplasmic reticulum on induction of apoptosis in fibroblasts.ResultsWe noted both qualitative and quantitative differences in the functional activity of these two anti-apoptotic proteins in cells: Bcl-2 localized to the endoplasmic reticulum inhibits apoptosis induced by ceramide and thapsigargin but not by doxorubicin or TNFα, while Bcl-XL at the endoplasmic reticulum is active against all four drugs. In fibroblasts Bcl-2 localized to the ER did not prevent cell death due to etoposide whereas Bcl-XL in the same location did. Finally in MCF-7 cells, Bcl-XL is approximately ten times more active than Bcl-2 in repressing apoptosis induced by doxorubicin. This difference can be manifest as a large difference in clonal survival.ConclusionWhen examined in the same cellular context, Bcl-2 and Bcl-XL differ substantially in the potency with which they inhibit apoptosis, mediated in part by differences in the inhibition of specific subcellular pathways.

tBid Undergoes Multiple Conformational Changes at the Membrane Required for Bax Activation

Background: tBid is a Bcl-2 family protein that promotes apoptosis at the mitochondria. Results: tBid undergoes a reversible conformational change at membranes before activation that is accelerated by Mtch2. Conclusion: The Mtch2 accelerated conformational change in membrane-bound tBid enables it to activate Bax. Significance: The conformational change in tBid is a novel potential site of apoptosis regulation. Bid is a Bcl-2 family protein that promotes apoptosis by activating Bax and eliciting mitochondrial outer membrane permeabilization (MOMP). Full-length Bid is cleaved in response to apoptotic stimuli into two fragments, p7 and tBid (p15), that are held together by strong hydrophobic interactions until the complex binds to membranes. The detailed mechanism(s) of fragment separation including tBid binding to membranes and release of the p7 fragment to the cytoplasm remain unclear. Using liposomes or isolated mitochondria with fluorescently labeled proteins at physiological concentrations as in vitro models, we report that the two components of the complex quickly separate upon interaction with a membrane. Once tBid binds to the membrane, it undergoes slow structural rearrangements that result in an equilibrium between two major tBid conformations on the membrane. The conformational change of tBid is a prerequisite for interaction with Bax and is, therefore, a novel step that can be modulated to promote or inhibit MOMP. Using automated high-throughput image analysis in cells, we show that down-regulation of Mtch2 causes a significant delay between tBid and Bax relocalization in cells. We propose that by promoting insertion of tBid via a conformational change at the mitochondrial outer membrane, Mtch2 accelerates tBid-mediated Bax activation and MOMP. Thus the interaction of Mtch2 and tBid is a potential target for therapeutic control of Bid initiated cell death.

Bax forms an oligomer via separate, yet interdependent, surfaces

Interactions of Bcl-2 family proteins regulate permeability of the mitochondrial outer membrane and apoptosis. In particular, Bax forms an oligomer that permeabilizes the membrane. To map the interface of the Bax oligomer we used Triton X-100 as a membrane surrogate and performed site-specific photocross-linking. Bax-specific adducts were formed through photo-reactive probes at multiple sites that can be grouped into two surfaces. The first surface overlaps with the BH1–3 groove formed by Bcl-2 Homology motif 1, 2, and 3; the second surface is a rear pocket located on the opposite side of the protein from the BH1–3 groove. Further cross-linking experiments using Bax BH3 peptides and mutants demonstrated that the two surfaces interact with their counterparts in neighboring proteins to form two separated interfaces and that interaction at the BH1–3 groove primes the rear pocket for further interaction. Therefore, Bax oligomerization proceeds through a series of interactions that occur at separate, yet allosterically, coupled interfaces.

Regulation of Ca2+-induced permeability transition by Bcl-2 is antagonized by Drp1 and hFis1

The regulation of mitochondrial permeability transition (MPT) is essential for cell survival. Un-controlled opening of the MPT pore is often associated with cell death. Anti-death protein Bcl-2 can block MPT as assessed by the enhanced capacity of mitochondria to accumulate and retain Ca2+. We report here that two proteins of the mitochondrial fission machinery, dynamin-related protein (Drp1) and human mitochondrial fission protein (hFis1), have an antagonistic effect on Bcl-2. Drp1, with the assistance of hFis1, sensitizes cells to MPT by reducing the mitochondrial Ca2+ retention capacity (CRC). While the reduction of CRC by Drp1/hFis1 is linked to mitochondrial fission, the antagonism between Bcl-2 and Drp1 appears to be mediated by mutually exclusive interactions of the two proteins with hFis1. The complexity of protein–protein interactions demonstrated in the present study suggests that in addition to the previously described role of Bcl-2 in the control of apoptosis, Bcl-2 may also participate directly or indirectly in the regulation of mitochondrial fission.

Suppression of IP3-mediated calcium release and apoptosis by Bcl-2 involves the participation of protein phosphatase 1

The involvement and potential interdependence of inositol trisphosphate (IP3) receptors and Bcl-2 in the regulation of Ca2+ signaling is not clear. Here, we have explored the mechanism(s) of how Bcl-2 suppresses the IP3-sensitive Ca2+ release in MCF-7 cells focusing on the possible role of protein phosphatase 1 (PP1). We found that through influences on protein–protein interaction, Bcl-2 may alter the balance between the effects of phosphatase (PP1) and kinase (PKA) on the IP3 R1 signaling complex. Using various experimental approaches including phosphatase inhibition and RNAi, we show that Bcl-2 by competing with IP3R1 for the binding of PP1 can reduce the IP3-mediated calcium signal and protect cells from mitochondrial dysfunction and cell death.

Multiple partners can kiss-and-run: Bax transfers between multiple membranes and permeabilizes those primed by tBid

During apoptosis Bid and Bax are sufficient for mitochondrial outer membrane permeabilization, releasing pro-apoptotic proteins such as cytochrome c and Smac/Diablo into the cytoplasm. In most cells, both Bid and Bax are cytoplasmic but bind to mitochondrial outer membranes to exert pro-apoptotic functions. Binding to membranes is regulated by cleavage of Bid to truncated Bid (tBid), by conformation changes in tBid and Bax, and by interactions with other proteins. At least at the peripherally bound stage, binding is reversible. Therefore, regulation of apoptosis is closely linked with the interactions of tBid and Bax with mitochondria. Here we use fluorescence techniques and cell-free systems containing mitochondria or liposomes that faithfully mimic tBid/Bax-dependent membrane permeabilization to study the dynamic interactions of the proteins with membranes. We confirm that the binding of both proteins to the membrane is reversible by quantifying the binding affinity of proteins for the membrane. For Bax, both peripherally bound (inactive) and oligomerized (active) proteins migrate between membranes but much slower than and independent of tBid. When re-localized to a new membrane, Bax inserts into and permeabilizes it only if primed by an activator. In the case of tBid, the process of transfer is synergetic with Bax in the sense that tBid ‘runs’ faster if it has been ‘kissed’ by Bax. Furthermore, Mtch2 accelerates the re-localization of tBid at the mitochondria. In contrast, binding to Bcl-XL dramatically impedes tBid re-localization by lowering the off-rate threefold. Our results suggest that the transfer of activated tBid and Bax to different mitochondria is governed by dynamic equilibria and potentially contributes more than previously anticipated to the dissemination of the permeabilization signal within the cell.